A Drosophila model of multiple endocrine neoplasia type 2

Abstract

Dominant mutations in the Ret receptor tyrosine kinase lead to the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN2). Mammalian tissue culture studies suggest that RetMEN2 mutations significantly alter Ret-signaling properties, but the precise mechanisms by which RetMEN2 promotes tumorigenesis remain poorly understood. To determine the signal transduction pathways required for RetMEN2 activity, we analyzed analogous mutations in the Drosophila Ret ortholog dRet. Overexpressed dRetMEN2 isoforms targeted to the developing retina led to aberrant cell proliferation, inappropriate cell fate specification, and excessive Ras pathway activation. Genetic analysis indicated that dRetMEN2 acts through the Ras-ERK, Src, and Jun kinase pathways. A genetic screen for mutations that dominantly suppress or enhance dRetMEN2 phenotypes identified new genes that are required for the phenotypic outcomes of dRetMEN2 activity. Finally, we identified human orthologs for many of these genes and examined their status in human tumors. Two of these loci showed loss of heterozygosity (LOH) within both sporadic and MEN2-associated pheochromocytomas, suggesting that they may contribute to Ret-dependent oncogenesis.

Figures

Figure 1.

Drosophila dRet is highly conserved compared to human Ret. (A) Detailed alignment of dRet (top sequence) and human Ret (bottom sequence) kinase domain and C terminus. Conserved kinase domain residues mutated in MEN2B patients are boxed in red; FMTC is boxed in blue. Conserved tyrosine residues are boxed in green; Y1015 in human Ret is the PLCγ-binding site (Borrello et al. 1996), human Y900 and Y905 are autoregulatory tyrosines in the activation loop that are required for RetMEN2A activity, and Y864 and Y952 are required for RetMEN2B activity (Iwashita et al. 1996, 1999). Y1062 in human Ret, highlighted by a green asterisk, is not strictly conserved in dRet, but the C-terminal tail of dRet is tyrosine rich (orange asterisks) and some of these tyrosines are in motifs that may be Grb2-binding sites. (B) Schematic of dRet protein structure. The cysteine-repeat region contains 14 cysteine residues. Residues mutated to create dRetMEN2A and dRetMEN2B are noted. (C–F) Larval dRet expression. (C and D) dRet expression within the larval brain and ventral nerve cord (vnc). Anterior is toward the right. (C) A low-magnification view. (D) A high-magnification view. Note the subset of strongly stained cells in the vnc (arrow) and the lighter-staining cells within the brain (arrow). (E and F) dRet expression within the third instar eye-antennal disc. (E) A low-magnification view. The eye field proper is the larger disc to the left; note the diffuse staining that indicates either low-level gene expression or background. (F) A high-magnification view of the dRet expressing developing ocelli (arrows), which are adjacent to the eye field.

Figure 2.

Overexpression of activated dRet causes a dramatic rough eye. dRetWT, dRetMEN2B (M955T), and dRetMEN2A (C695R) overexpressed in the retina from the GMR promoter. (B, D, and F) Phenotypes conferred by one copy of a GMR-Ret transgene, dRet isoform indicated. (C, E, and G) Phenotypes conferred by two copies of each GMR-dRet transgene.

Figure 3.

GMR-dRetMEN2B directs excess proliferation, patterning defects, excess neuronal differentiation, and ectopic ERK activation. Anterior is toward the right. (A and B) The larval eye field differentiates in a posterior-to-anterior wave, and the brackets denote the second wave of mitoses; S-phase nuclei are visualized with BrdU (orange); note the ectopic S-phase cells in GMR-dRetMEN2B tissue. (C and D) Eye discs from GMR-dRetMEN2B flies (two copies) display a normal pattern of Boss staining (orange), indicating that early photoreceptor development (R8) is normal. Staining for Atonal (another marker for R8) in GMR-dRetMEN2B tissue is also normal (data not shown). These stains show that early development proceeds normally before GMR-driven dRet expression begins and that R8 retains its normal identity. (E and F) Eye discs from GMR-dRetMEN2B flies (two copies) contain disorganized Bar-expressing cells (arrows in F), suggesting aberrant recruitment, specification, or patterning of later-developing photoreceptor cells R1 and R6. (G and H) Eye tissue from GMR-dRetMEN2B flies (two copies) stained for Sev protein (orange) shows ectopic and aberrant clusters of Sev-expressing cells (arrows in H). Note that older, more posterior clusters of Sev-expressing cells are improperly spaced. (I and J) Photoreceptor neurons are visualized with 22C10, a neuronal-specific antibody. GMR-dRetMEN2B (two copies) eye tissue contains ectopic neurons (arrows in J) between abnormally spaced ommatidia containing variable numbers of neurons. (K and L) An antibody specific for activated, di-phospho-ERK protein indicated that GMR-dRetMEN2B (two copies) eye tissue contains high levels of activated ERK in irregular patches (bracket in L) posterior to the morphogenetic furrow after the onset of GMR-induced expression. Early di-phospho-ERK staining (anterior to bracket in L) is normal in GMR-dRetMEN2B discs prior to the onset of GMR-induced expression.

Figure 4.

Abnormal patterning and differentiation of ommatidia and interommatidial lattice cells in more mature GMR-dRetMEN2B retina. (A and B) Forty-two-hour pupal eye imaginal discs; apical profiles of cells are visualized with an antibody specific for the junctional protein Armadillo. (A) A wild-type pupal eye showing approximately a dozen ommatidia. Cells within and surrounding one ommatidium are labeled to indicate cone cells (c) and primary (1), secondary (2), and tertiary (3) pigment cells, and bristles (b) define an interommatidial lattice. (B) A single copy of GMR-dRetMEN2B resulted in a milder phenotype that included abnormal numbers of cone cells and a poorly patterned interommatidial lattice. (C) GMR-dRetMEN2B (two copies) eyes show a marked lack of ommatidial organization including an absence of clearly definable cells. The rounded cuboidal shape of these cells is typically seen in undifferentiated lattice cells early in normal pupal retinal development. (D) Plastic sections from adult eye tissue. Wild type (inset) has a normal complement of seven photoreceptor neurons within each ommatidium; they are most easily seen by their rhabdomeres, which appear as solid blue circles in the section. Note that each rhabdomere array forms a stereotyped trapezoid that “points” upward (arrows in inset). GMR-dRetMEN2B (two copies) sections contain abnormally assembled and patterned ommatidia and large vacuolated spaces.

Figure 5.

Enhancer-suppressor screen for modifiers of GMR-dRetMEN2. (A) Examples of GMR-dRetMEN2B modifier phenotypes, which were scored using a dissecting microscope. Enhanced flies had a worsened phenotype and suppressed flies had a milder phenotype. (B) Diagram of parental (P) genetic crosses and F1 progeny genotypes. GMR-dRetMEN2B, GMR-dRetMEN2A, and GMR-dRetWT flies were crossed to flies heterozygous for loss-of-function deficiency mutations; each mutation was maintained over a wild-type balancer chromosome (balancer chromosomes contain breakpoints that prevent meiotic recombination and carry visible markers, but are otherwise genetically wild type and are denoted as such here). The phenotypes of different classes of progeny were compared and the penetrance was calculated for any observed genetic interactions.